Heterologous expression of human cytochrome

P450 (CYP) 2C19 in Escherichia coli and

establishment of RP-HPLC method to
serve as activity marker
Yan Pan
a
*, Joon Wah Mak
a
and Chin Eng Ong
b
ABSTRACT: In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method
was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This
system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli)
cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral
scanning. The RP-HPLC assay showed good linearity (r
2
= 1.00) with 4-hydroxymephenytoin concentration from 0.100 to
50.0 mM and the limit of detection was 5.00 10
2
mM. Intraday and interday precisions determined were from 1.90 to
8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0
to 105%. Enzyme kinetic parameters (K
m
, V
max
and K
i
) were comparable to reported values. The presence of CYP2C19 in
bacterial membranes was conrmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined
in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to
that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the
RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
Copyright 2013 John Wiley & Sons, Ltd.
Keywords: HPLC; CYP2C19; in vitro; drug interaction
Introduction
Cytochrome P450 (CYP) is a superfamily of membrane-bound
heme proteins with characteristic absorption peak at 450 nm
when reduced by CO. CYPs function as mono-oxygenases
catalyzing numerous endogenous and exogenous substances.
A signicant number of clinically used drugs also undergo
metabolism mediated by CYPs, and mainly CYP1, 2 and 3 families
are involved. Concurrent intake of CYP substrates, inhibitors or
inducers increases the risk of adverse drug reactions owing to
drugdrug interactions. CYP2C19 belongs to the CYP2C subfamily
and is responsible for metabolizing prescribed drugs, including
omeprazole (Andersson et al., 1992), proguanil (Ward et al., 1991),
certain barbiturates (Adedoyin et al., 1994), citalopram (Sindrup
et al., 1993) and diazepam (Bertilsson et al., 1993). This isoform
receives special attention with regard to its genetic polymorphism,
with ethnic differences in poor metabolizer frequency, rang-
ing from 25% in Caucasians to 1823% in Asian population
(Nakamura et al., 1985; Wilkinson et al., 1989).
In vitro screening of drugdrug interaction potentials is always
suggested before performing in vivo tests using human subjects.
One important screening parameter is modulation of CYP cata-
lytic activity. In order to determine the alteration of CYP activity,
several types of enzyme sources are employed, including human
liver microsomes, liver homogenates and hepatocytes. However,
problems such as short supply of the tissue sources, ethical
concerns in relation to patient consents and inconsistencies of
population of CYP isoforms from different donors limit their use.
Moreover, the inherent low catalytic activity of CYP2C19 is
another potential problem in using human-derived enzymes.
Heterologous expression of human CYPs has become an alterna-
tive enzyme source in in vitro predictions on drugdrug interac-
tions. Human CYPs expressed from mammalian cells (Hyland
et al., 2001), yeast (Eugster et al., 1990) and bacteria (Larson
et al., 1991) have demonstrated sufcient activities in in vitro stud-
ies. Among these systems, the E. coli system has become one of
the popular choices owing to its lower cost of maintenance, ease
of use and high yield of protein within a relatively short period of
culture time. Nevertheless, to achieve a high expression level in
bacterial systems, native human CYP cDNA sequences need to
be modied to overcome species differences of codon prefer-
ences between mammals and bacteria (Waterman, 1993). The rst
catalytically active CYP17a protein was expressed in E. coli cells
after N-terminal modication (Barnes et al., 1991). Subsequently
* Correspondence to: Y. Pan, School of Medical Sciences, International
Medical University, 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur,
Malaysia. Email: panyan1980@hotmail.com
a
School of Medical Sciences, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000
Kuala Lumpur, Malaysia
b
Jeffrey Cheah School of Medicine and Health Sciences, Monash University
Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor
Darul Ehsan, Malaysia
Abbreviation used: CYP, cytochrome P450; OxR, oxidoreductase
Biomed. Chromatogr. 2013; 27: 859865 Copyright 2013 John Wiley & Sons, Ltd.
Research article
Received: 3 September 2012, Revised: 19 December 2012, Accepted: 24 December 2012 Published online in Wiley Online Library: 6 February 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.2872
8
5
9
many human CYP isoforms have been expressed with the same
modications and used in in vitro assays.
S-Mephenytoin is a hydantoin, used as an anticonvulsant.
Its use as anticonvulsant, however, is obsolete as there are
many other safer and more effective antiepileptic agents
available. CYP2C19 was identied as the major CYP isoform
that metabolizes S-mephenytoin 4-hydroxylation in humans;
hence, S-mephenytoin has been commonly used as activity
marker for CYP2C19 (Goldstein and Faletto, 1994). Here, we
have described a procedure to co-express functional active
human CYP2C19 and OxR from E. coli cells and the enzyme
activity was characterized by a RP-HPLC assay, which was devel-
oped and validated in the present study as well.
Experimental
Chemicals and materials
Acetonitrile and dichloromethane (all HPLC-grade) were purchased from
Fisher Scientic (Pittsburgh, PA, USA). The other chemicals [S-mephenytoin,
4-hydroxymephenytoin, phenytoin, omeprazole, b-nicotinamide adenine
dinucleotide phosphate (NADP), D-glucose 6-phosphate (G-6-P), glucose-
6-phosphate dehydrogenase and magnesium chloride] were all purchased
from Sigma (St Louis, MO, USA) with the highest grades available.
Construction of expression plasmids
Native CYP2C19 cDNA was generated by reverse-transcriptase polymeras
chain reaction (RT-PCR) method and subcloned in the plasmid Bluescript
W
II SK + (pBS-2C19) by previous work in our laboratory (data unpublished;
Pan et al., 2011). Bacterial expression vector pCWori+ and plasmid
pACYC-ompA-OxR were kindly provided by Professors John Miners and
Donald Birkett (Flinders University, Adelaide, Australia). 17a Sequence,
MALLLAVFL, was introduced into the N-terminus of the CYP2C19 cDNA
by PCR-based mutagenesis with the help of a mutagenic primer contain-
ing an NdeI site and a reverse primer completely complementary to a
HindIII site located just outside CYP2C19 cDNA stop codon (Table 1). The
PCR product and pCWori+ vector were digested with NdeI and HindIII and
then ligated to form pCW-2C19. The general scheme is illustrated in Fig. 1.
DNA sequencing of both strands at full length of the constructed
pCW-2C19 was performedto ensure that no errors had been introduceddur-
ing the amplication process. The veried plasmid was then co-transformed
with pACYC-ompA-OxR into an E. coli DH5a bacterial cell. Successfully
co-transformed cells were prepared and kept as 70.0% glycerol stocks in
80

C freezer. The cDNA for CYP2C19 were expressed in E. coli cells.

Expression and purication of the proteins were carried out according to
an established protocol in our laboratory (Singh et al., 2008).
Quantication of recombinant protein expression
The CYP2C19 spectral content of the bacterial cell lysates was quantied
using a dual-wavelength/double beam spectrophotometer (Shimadzu,
Tokyo, Japan). This spectrophotometer also allowed us to estimate the
OxR activity using cytochrome c as a substitute electron acceptor. Both
experimental procedures were described in detail in our previously
published paper (Pan et al., 2011).
Immunoblotting of expressed CYP2C19 protein
The expression of CYP2C19 in the E. coli whole cells was demonstrated
by immunoblotting analysis. CYP2C19 protein was analyzed using SDS
PAGE in 12.0% polyacrylamide gel based on Laemmlis (1970) method.
After running SDS-PAGE, the separated proteins were transferred to a
nitrocellulose membrane. Primary antibody (rabbit antihuman CYP2C8/
9/19 polyclonal antibody), which is able to bind CYP2C19 protein, was
incubated with the nitrocellulose membrane followed by incubation
with horseradish peroxidase-conjugated secondary antibody (polyclonal
goat anti rabbit antibody). The presence of CYP protein was visualized
using a chloronapthol developing solution (containing 15.0 mg of
4-cholro-1-napthol, 5.00 mL of ethanol, 50.0 mL of Tris-buffered saline
and 50.0 mL of 30.0% hydrogen peroxide).
Table 1. Sequences of oligonucleotide primers used for PCR
mutagenesis of the CYP2C19 N-terminus
CYP form Sequence of oligonucleotide primers
CYP2C19 Forward primer (NdeI
a
)
M A L L
5
0
a gga att cat atg gct ctg tta
L A V F L
tta gca gtt ttt ctg tgt ctc tca tgt
ttg ctt ctc 3
0
Reverse primer (HindIII
a
)
5
0
agg gaa ttc aag ctt tca gac agg aat gaa gca
cag ctg 3
0
a
Restriction enzyme cutting sites are in bold type in the
sequences.
NdeI HindIII
+
T4 ligase
+ pCW ori+
AmpR
AmpR
pCW ori+
pBS-2C19
+
Forward primer
Reverse primer
Taq
PCR
NdeI HindIII
PCR-2C19
AmpR
pCW-2C19
PCR template
Figure 1. General scheme illustrating the process of N-terminal modication of CYP2C19 cDNA to generate pCW-2C19 construct. The template used in
the PCR was pBS-2C19 where CYP2C19 native cDNA was subcloned in the plasmid Bluescript
W
II SK+ by previous work (data unpublished).
Y. Pan et al.
Biomed. Chromatogr. 2013; 27: 859865 Copyright 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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RP-HPLC conditions
The RP-HPLC assay for determining 4-hydroxymephenytoin was per-
formed according to the published method with minor modications
(Ko et al., 2000) employing a Gilson HPLC system, which consisted of a
Gilson 307 pump, a Gilson UV/vis-152 detector and an ODS HYPERSIL
column (Thermo, 4.6 250 mm, 5 mm). The mobile phase consisted of
26.0% acetonitrile (in 0.0500 M phosphate buffer, pH 5.00) and was
pumped at a ow rate of 1.50 mL/min at wavelength of 211 nm. The
whole operation lasted 25 min. A sample volume of 50.0 mL of the
solution was subjected to HPLC analysis.
Calibration curve
The calibration curve was established by injecting different concentrations
of 4-hydroxymephenytoin (0.10050.0 mM) prepared from stock solution
onto the HPLC system. The peak area ratios of 4-hydroxymephenytoin to
phenytoin (internal standard) were calculated and used for constructing
calibration curves.
Method validation
The RP-HPLC assay mentioned in this study was validated for the follow-
ing parameters: solution stability, specicity, linearity, detection limit,
precision, accuracy and recovery.
Stock and sample solution stability. The stock solutions of
S-mephenytoin and 4-hydroxymephenytoin were prepared at 50.0 mM in
acetonitrile. Phenytoin (internal standard) was prepared at 0.100 g/mL in
methanol. All stock solutions were stored at 20

C, and were found

to be stable for at least a month. Solution stability of all analytes
(S-mephenytoin, 4-hydroxymephenytoin and phenytoin) after sample
collection and preparation were found to be stable for up to 48 h. All
our samples were analyzed by being injected onto HPLC system within
12 h of preparations on routine basis.
Specicity of the assay condition. The specicity of the method de-
veloped was ascertained by comparing the separation of S-mephenytoin,
4-hydroxymephenytoin and phenytoin in sample incubation (containing
CYP2C19 protein) with control incubation (containing control protein,
bacterial membranes isolated from culture stock transformed with
pCWori+ plasmid with no CYP2C19 cDNA). Triplicates of comparison
were carried out to demonstrate the lack of endogenous interference
and batch-to-batch variation.
Linearity and limit of detection. Linear regression analysis was per-
formed to determine the slope, intercept and r
2
value of calibration
curves obtained at seven 4-hydroxymephenytoin concentration levels
(0.10050.0 mM). The limit of detection (LOD) was estimated at a signal-
to-noise ratio of 3:1 by injecting a series of diluted solutions with
known concentrations of 4-hydroxymephenytoin (2.00 10
3
, 5.00
10
3
, 1.00 10
2
, 2.00 10
2
and 5.00 10
2
mM).
Precision. Intra- and inter-day coefcients of variation (% CV) were
determined by measuring the spiked 4-hydroxymephenytoin concen-
trations in incubation mixtures. This was performed by measuring the
peak area ratios (4-hydroxymephenytoin over phenytoin), which was
injected in three concentrations, namely 0.500, 5.00 and 20.0 mM.
Each of these concentrations was injected in triplicates onto the
system on the same day to obtain intraday precision. Interday pre-
cision determination involved injection of the same concentrations
on three consecutive days. The data obtained were calculated as
repeatability of recovered amounts, expressed by mean, standard
deviation (SD), and coefcient of variation (% CV = SD/mean for each
4-hydroxymephenytoin concentration).
Accuracy. Accuracy was determined by comparing the estimated
amount with the known 4-hydroxymephenytoin concentration of spiked
samples at 0.500, 5.00 and 20.0 mM in triplicate. The results were then
expressed as the percentage of bias between the nominal and the calcu-
lated concentrations.
Recovery. Recovery of 4-hydroxymephenytoin was determined by
comparing peak area ratios of 4-hydroxymephenytoin to phenytoin
from extracted incubation mixtures, with those from equivalent
amounts of 4-hydroxymephenytoin spiked directly into extraction
solvent (dichloromethane). The recovery was determined by comparing
the calculated concentration with the known 4-hydroxymephenytoin
concentration of spiked samples at concentrations of 0.500, 5.00 and
20.0 mM three times. The results were then expressed as the percentage
of bias of determined concentrations between the incubation and the
spiked mixtures.
CYP2C19-mediated S-mephenytoin 4-hydroxylase assay
Basically, reactions were initiated by the addition of membrane protein
and were carried out in air at 37

C in a metabolic shaker for 60 min. Each

reaction mixture (250 mL) contained bacterial membrane protein, an
NADPH generating system (1.00 mM NADP, 10.0 mM G-6-P, 2.00 IU G-6-P
dehydrogenase and 5.00 mM MgCl
2
) in 0.100 M phosphate buffer, pH
7.400 and S-mephenytoin. The reaction was terminated by addition of
130 mL of acetonitrile (containing 30.0 mL of 20.0 mg/mL phenytoin as inter-
nal standard) and cooling on ice followed by extraction with 1.00 mL of
dichloromethane via votexing. The organic layer was then collected and
dried. The residue was dissolved in 120 mL of mobile phase, and 50.0 mL
of the solution was subjected to HPLC analysis.
Protein and time curves
The effects of protein concentration and incubation time were examined
by constructing the protein and time curves. Protein curve was gener-
ated by incubating a series of concentrations (0.2004.00 mg/mL) of
expressed CYP2C19 with S-mephenytoin at the nal concentration of
100 mM at 37

C for 60 min. The time curve was constructed by incubating

0.200 mg/mL CYP2C19 with S-mephenytoin (nal concentration 100 mM)
at 37

C for various periods (10180 min).

Kinetic characterization of CYP2C19
In order to characterize the catalytic activity of the CYP2C19, S-mephenytoin
was incubated in various concentrations (ranging from1.00 to 1.00 10
3
mM)
with 5.50 10
2
mg CYP2C19 protein in volumes of 250 mL for 60 min.
Using these data, saturation and EadieHofstee plots were created and
K
m
and V
max
values were determined with the EZ-t
W
kinetic software
(Perrella Scientic Inc., Amherst, NH, USA).
Results and discussion
Heterologous expression of CYP2C19 in E. coli
In this study, sequence (MALLLAVFL) was added to the N-terminus
of CYP2C19 cDNA and ligated to pCWori+ plasmid, which was
subsequently transformed into E. coli DH5a cells for expression.
This modication strategy was adopted to overcome low expres-
sion level of human CYP protein achieved by bacteria owing to
species difference in the preferred codon. Moreover, the E. coli
cells do not possess an endogenous electron transport system
to support the full catalytic activity of CYP enzymes; co-expression
of CYP and OxR in E. coli cells has been found to be necessary to
achieve efcient catalytic ability. We have reported co-expressed
pCW-2D6 or pCW-3A4 with pACYC-OxR as separate proteins,
demonstrating satisfactory expression levels and catalytic activi-
ties (Pan et al., 2011). Here we employed the same strategy to
co-express CYP2C19 and OxR for use in the subsequent study.
Expression of CYP2C19 and establishment of RP-HPLC method
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pCW-2C19 and pACYC-ompA-OxR were successfully co-expressed
from E. coli DH5a cells. The yield of membrane protein following
routine culture protocol was high with values of 21.0 3.62mg
membrane protein per litre culture. OxR expression level was
980 143 nmol/min/mg membrane determined fromcytochrome
c reductase assay, which fell within the reported range in the litera-
tures (Blake et al., 1996; Pritchard et al., 1998) and these showed
sufcient activities, allowing optimal activity for CYPs in oxidizing
various substrates. The harvested and fractionated membrane
proteins were analyzed using western immunoblotting (Fig. 2),
showing a band with a molecular mass of approximately 50 kDa,
which was in accordance with the size reported in the literature
(Schulz-Utermoehl et al., 2000a, 2000b). In the meantime, control
protein expressed from DH5a cells transformed with pCWori+
plasmid without any foreign gene did not demonstrate detectable
band at this site. Nevertheless, the western blotting result only
revealed the level of full-length total protein but not the type of
the protein (whether they were apoprotein or holoprotein). Hence,
reduced CO difference spectral scanning was carried out, which
showed an absorbance maximumat 450 nmwhile the control cells
that were transformed with the blank pCWori+ vector did not
show any absorbance peak (Fig. 3). This indicated the successful
expression of functional protein (i.e. in the form of holoenzyme).
Our result once again demonstrated that E. coli expression system
constructed with co-transforming OxR and CYP was able to
obtain high output capacity. More than one foreign protein was
expressed from DH5a cells with negligible effects with regard to
competition between the genes of interest for the transcriptional
and translational machinery of the cells. The result obtained is in
line with ndings from other researchers as well as our previous
study, where high levels of OxR and CYP proteins were simulta-
neously achieved (Gillam 1998; Pan et al., 2011).
HPLC method development
Calibration curve, linearity and limit of detection. The line-
arity determined by linear regression analysis was found to be
quite satisfactory and reproducible with time. The equation of
mean linear regression (n = 3) was y = 5.82 10
2
x + 1.23
10
2
with an r
2
value of 1.00. Sequentially diluted solutions of
4-hydroxymephenytoin injected directly onto the HPLC system
revealed that 5.00 10
2
mM was the LOD for this assay.
Specicity. Figure 4 shows representative chromatograms
obtained from incubation mixtures containing the expressed
CYP2C19 and OxR in comparison to that of the control cell.
34.3KD
50KD
103KD
77KD
1 2 3
28.8KD
20.7KD
Figure 2. Immunoblotting analyses of CYP2C19 expression in DH5a
cells. The E. coli membrane proteins (150 mg) were electrophoresed on
a 12.0% SDSpolyacrylamide gel, transferred to a nitrocellulose lter
and reacted with appropriateantibody to CYP2C19. The labeled proteins
were developed using peroxidase-conjugated goat anti-rabbit IgG. Lane 1,
molecular weight markers (prestained protein standards, low range, Bio-
Rad, USA); lane 2, membrane fractions of the control cell carrying blank
pCWori+ plasmid; lane 3, membrane fractions isolated from DH5a culture
stocks carrying the co-transformed OxR and CYP2C19 plasmids.
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
400 420 440 460 480 500
A
b
s
o
r
b
a
n
c
e
Wavelength (nm)
Figure 3. Reduced COdifference spectra showing expression of CYP2C19
(solid line) and the control (dotted line). The arrow indicates the absor-
bance peak at 450 nm wavelength.
(B)
1
2
3
2
1
(A)
Figure 4. Representative HPLC chromatograms of incubations of
S-mephenytoin (100 mM) with (A) 5.50 10
2
mg/250 mL control cell
membranes at 37

C for 60 min. Peak 1, S-mephenytoin (elution time =

12.5 min); peak 2, internal standard (elution time = 22.5 min); (B) 5.50
10
2
mg/250 mL bacterial membranes (expressing CYP2C19 and OxR) at
37

C for 60 min. Peak 3, Hydroxymephenytoin (elution time = 4.8 min);

peak 1, S-mephenytoin (elution time = 12.5 min); peak 2, internal standard
(elution time = 22.5 min).
Y. Pan et al.
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As shown in the gure, the peaks for 4-hydroxymephenytoin
(the metabolite), S-mephenytoin and phenytoin (internal stan-
dard), eluted at retention times of 4.8, 12.5 and 22.5 min, respec-
tively, exhibited adequate symmetry and were well separated.
No detectable 4-hydroxymephenytoin peak was observed in
the control incubations. The metabolite peak was proven to be
that of 4-hydroxymephenytoin as injection of pure metabolite
onto the system yielded a peak with the same retention time.
Precision. The intra- and interday precisions of 4-hydroxyme-
phenytoin were obtained at three different concentrations of
0.500, 5.00 and 20.0 mM. The intraday precision ranged from
1.90 to 8.19% while interday precision ranged from 2.20 to
14.9%, which fell within the limit (i.e. below the upper limit of
15.0%) usually accepted for HPLC assay validation, indicating
that the method developed has allowed precise and reliable
measurement of 4-hydroxymephenytoin within the concentra-
tion range investigated.
Accuracy and recovery. The accuracies determined using
standard 4-hydroxymephenytoin at concentrations of 0.50, 5.00
and 20.0 mM were 101 4.30%, 95.0 6.20% and 105 15.9%,
respectively. The calculated recoveries were 83.5 6.30%
(0.500 mM), 85.8 14.8% (5.00 mM) and 85.2 2.50% (20.0 mM).
Both results fell within acceptable range (80.0120%). Thus, no
signicant difference was found between calculated concentra-
tions and known concentrations while no signicant differences
were also noted between extracted samples with directly spiked
samples, indicating that the method developed was accurate
enough and the extraction procedure did not affect the assay.
Protein and time curves. The linear range for expressed
CYP2C19 protein was from 0.200 to 0.800 mg/mL. The reaction
rate stabilized thereafter at higher protein concentrations. The
curve also shows that the linear range for incubation time was
from 10 to approximately 120 min, after which the velocity curve
started to level and stabilize. Hence 0.220 mg/mL bacterial
membrane protein was incubated for 60min for subsequent
routine reactions.
Kinetics of CYP2C19-mediatedS-mephenytoin4-hydroxylation
Figure 5(A) shows that 4-hydroxymephenytoin formation fol-
lowed MichaelisMenten kinetics {v = V
max
[S]/(K
m
+[S])}, where
the velocity (v) of the reaction follows a hyperbolic pattern and
approaches the maximum velocity (V
max
) with increasing sub-
strate concentrations ([S]) and K
m
is the substrate concentration
at which the reaction rate is half of its maximum. Similarly the
EadieHofstee plot (Fig. 5B) exhibited a straight line, indicating
involvement of a single enzyme in the incubation reactions,
which is expected as CYP2C19 was the only enzyme expressed
in the bacterial membranes. The K
m
value determined from the
plot, 108 9.20 mM, resides close to and within the variability
of reported values for CYP2C19 in the literature (Table 2), indicat-
ing that the expressed CYP2C19 from this study was as active as
those reported by others. However, there was a big difference
between the V
max
(1.20 10
3
18.4 pmol/min/mg protein)
determined from this study and reported values (Table 2). There
will undoubtedly be differences between reports of V
max
values
in human liver microsomes and various cDNA-expressed proteins
Saturation Plot
v
S
0
500
1000
1500
2000
0 500 1000 1500 2000 2500 3000 3500 4000 4500
0
200
400
600
800
1000
0 5 10 15
(A) (B)
V/S
Eadie-Hofstee Plot
V
V
Figure 5. Representative velocities vs substrate concentrations plot (A) and EadieHofstee plot (B) for hydroxymephenytoin formation in DH5a mem-
branes expressing the CYP2C19. Points are experimentally determined values while solid lines are the computer-generated curves of the best t. V,
Velocity in pmol/min/mg; S, S-mephenytoin concentration in (mM).
Table 2. K
m
and V
max
values collected from literatures for CYP2C19 mediated S-mephenytoin 4-hydroxylation
Report K
m
(mM) V
max
(pmol/min/mg protein) Reference
1 57.2 2.20
a
58.3 0.800
a
Walsky and Obach (2004)
2 35.0 7.00
a
20.0 1.00
a
Di Marco et al. (2007)
3 23.1 69.4 Li et al. (2003)
4 72.4 40.4
a
70.5 48.0
a
Wrighton et al. (1993)
5 52.7 55.0 2.00
a
Hickman et al. (1998)
6 59.0143 12.7-80.8 Meier et al. (1985)
a
Data are represented as means SD.
Expression of CYP2C19 and establishment of RP-HPLC method
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in heterologous expression system because these values are a
function of the level of expression of enzyme in different sys-
tem and the incubation conditions adopted in different labora-
tories. On the other hand, K
m
values should not differ greatly
from preparation to preparation and should be comparable
from one report to the next, since the parameter is an inherent
property of the enzyme and should only potentially differ
when the amino acid sequences of the enzymes show genetic
variation, or if the activity being measured is not highly selec-
tive for one enzyme and has varying contributions from other
enzymes. Nevertheless, it should be noted that K
m
values mea-
sured for the same reaction using different CYP2C19 sources
still demonstrated some differences especially between pooled
human liver microsomes and recombinant enzymes. Such dif-
ferences are not uncommon and a number of factors could
contribute to this variability. These include differences in pro-
tein concentrations used in vitro, differences in ratios of OxR
and/or cytochrome b5 vs CYP in protein preparations, and
differences in phospholipid composition of microsomes from
expression systems vs that of liver tissue.
Conclusion
As a conclusion, N-terminal modication introduced into CYP2C19
cDNA has allowed successful co-expression of CYP2C19 protein
together with OxR protein in E. coli DH5a cells. This was judged
by immunoblotting and reduced CO difference spectral assay,
as well as cytochrome c reductase assay. In this work, we also
developed and validated an RP-HPLC assay to determine the
amount of 4-hydroxymephenytoin, which met the criteria for
various validation characteristics for analytical procedures,
including specicity, linearity, lower limit of detection, preci-
sion, accuracy and recovery. This validated assay was applied
to characterize kinetics for CYP2C19 by incubating expressed
protein with S-mephenytoin and co-factors. Enzyme kinetic
parameters obtained demonstrated values that were either
close to or within the range of values previously reported in
literatures. In a nutshell, the protein expression strategy and
the validated HPLC method described in this study were able
to serve as activity markers for investigating drug metabolism
and interactions involving CYP2C19.
Acknowledgments
The authors are thankful to the International Medical University,
Malaysia (grant IMU 091-05), as well as the Malaysian Ministry of
Science, Technology and Innovation (grant eScienceFund 02-02-
09-SF0005) for nancial support.
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